The predictable and safe induction of human organ transplant tolerance remains an elusive, much sought-after goal. In most patients, acute rejection can be managed effectively, but many allografts succumb to the ravages of chronic rejection. Bone marrow-derived dendritic cells (DC) are uniquely well-equipped antigen (Ag)-presenting cells, now regarded as critical regulators of immune reactivity. Immature/semi-mature DC, that express low levels of costimulatory molecules necessary for the induction of effector T cell responses, have inherent tolerogenic potential. These cells are a promising means of improving transplant outcome. The challenge is to identify the maturation-resistant DC best-suited to promote therapeutic tolerance. Our recent data show that rapamycin (RAPA), an immunophilin- binding immunosuppressive pro-drug, well-recognized for its inhibitory action on T cell growth, stably suppresses DC maturation, inhibiting their responses to Toll-receptor and CD40 ligation and pro-inflammatory cytokines. These RAPA- treated DC (RAPA-DC) induce alloAg-specific T cell hyporesponsiveness in vitro and in vivo and expand CD4+CD25*Foxp3+ T regulatory cells (Treg) in vitro. Furthermore, pre-operative infusion of MHC-mismatched organ graft recipients with host-derived RAPA-DC, pulsed with donor Ag, promotes donor-specific graft survival. Repeated infusion, or a short course of minimal immunosuppression, enhances long-term transplant survival, associated with intra-graft Treg. Based on these encouraging data, we hypothesize that a regimen based on treatment of allograft recipients with recipient-derived, pharmacologically-modified, maturation-resistant DC (RAPA-DC) pulsed with donor Ag, and capable of impacting the indirect pathway of allorecognition, will promote stable long-term allograft survival. We further hypothesize that this approach will lead to development of immune regulation via T reg. Based on emerging evidence of the role of autoimmunity to tissue-specific Ag in organ graft rejection, we will also explore the novel concept that pulsing of RAPA-DC with tissue-specific Ag may enhance their tolerogenicity. We have four specific Aims. In AIM I, we will elucidate molecular mechanisms underlying the inhibitory action of RAPA on DC maturation and optimize generation of maturation-resistant murine DC in vitro. We will assess how RAPA affects signaling via Toll and cytokine receptors to maximize their tolerogenic potential;in AIM II, we will determine the optimal protocol for induction of long-term, rejection-free, organ allograft survival using RAPA-DC, including the use of co-stimulation blockade. In AIM III, we will ascertain mechanisms underlying alloAg presentation and regulation of alloAg-specific T cell responses by RAPA-DC focusing on the role of T reg. In AIM IV, we will generate maturation-resistant human RAPA-DC for potential clinical application. These studies will pave the way for the future use of DC as therapeutic vectors of transplant tolerance. Manipulation of dendritic cells to better control organ transplant rejection and to reduce patients'dependency on anti-rejection drugs, promises to reduce morbidity and mortality in the transplant population and to minimize the attrition